SEQUENCING OF BACTERIAL AND ARCHAEAL 16S RIBOSOMAL-RNA GENES DIRECTLYAMPLIFIED FROM A HYPERSALINE ENVIRONMENT

The new methods based on PCR amplification and sequencing of 16S rRNA genes from DNA samples extracted directly from the environment allow t he description of microbial diversity in natural ecosystems without th e need for cultivation. We have applied this technique to an extreme e nvironment presumed to have very low diversity: the crystallizer ponds of a marine saltern (salinity over NaCl saturation). The molecular me thodology has shown that indeed very low diversity can be found here. Bacteria (formerly eubacteria) are largely minoritary, and only a clus ter of closely related sequences was found, all distant relatives of t he alpha-Proteobacteria (82% to Rhodopseudomonas marina). Halophilic A rchaea were shown by hybridization to be, as expected, the largest com ponent of biomass in this environment. All the archaeal clones sequenc ed again were highly similar to each other suggesting that they are pr obably members of the same genus. However, all the sequences diverged considerably from those of the described genera of halophilic Archaea. In fact the data are consistent with the idea that the 16S rDNA genes directly amplified from the saltern correspond to members of an undes cribed genus which seems to be abundant in the sample. This is remarka ble since many collection strains sequenced come specifically from thi s same saltern. Furthermore, 16S rDNA obtained from archaeal cultures isolated from the same sample had no homology to the sequences obtaine d by PCR amplification, instead they appear to be members of the well known genus Haloarcula. However, this is in agreement with the finding s of other authors who by culture have obtained organisms different fr om those indicated by the sequences retrieved directly by PCR. Possibl e explanations are discussed.